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  • C12N9/10—Transferases (2.)
  • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
  • C12N9/1007—Methyltransferases (general) (2.1.1.)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to the use of DNA damaging agents for the treatment of cancer.
• DNA damaging agents such as doxorubicin
• DNA damaging agents have been widely used in the treatment of cancer.
• Such agents selectively kill proliferating cells while being less toxic to non-proliferating cells, thus providing some measure of cancer cell selectivity, since most cells of the body are non-proliferating.
• important normal cell types such as intestinal endothelium, immune system cells, bone marrow cells and hair follicle cells do proliferate, and thus are also killed by DNA damaging agents, leading to numerous unwanted side effects. There is, therefore a need to improve the efficacy and selectivity of DNA damaging agents for the treatment of cancer.
• the invention relates to the treatment of cancer using DNA damaging agents.
• the inventor has surprisingly discovered that knockdown of a previously uncharacterized gene, acidic residue methyltransferase (Arml), improves the ability of cells having a wild-type p53 gene to survive treatment with DNA damaging agents, while causing cells having mutant p53 genes to become more sensitive to killing by DNA damaging agents. Since more than 50% of cancer cell types have mutant p53 genes, while normal proliferating cells have wild type p53 genes, inhibition of Arml increases both the efficacy and selectivity of DNA damaging agents for killing cancer cells.
• Arml acidic residue methyltransferase
• the invention provides a method for treating a mammal with cancer, the method comprising inhibiting in the mammal acidic residue methyltransferase (Arml) in combination with administering to the mammal a DNA damaging agent.
• Arml acidic residue methyltransferase
• the invention provides a pharmaceutical formulation comprising an inhibitor of acidic residue methyltransferase (Arml) and a DNA damaging agent.
• Arml acidic residue methyltransferase
• Figure 1 shows that C6orf211 encodes a PCNA-dependent carboxyl
• methyltransferase (Arml).
• a A carboxyl methyltransferase targets PCNA in MDA MB 468 cells
• b SAM-dependent methyltransferase domains exist in the C6orf211 protein
• c The positions of motifs I, II and regions II and III in CheR and the C6orf211 protein
• d Illustrative representation of the SAM-MT fold in the C-terminus of the CheR.
• e The C- terminus of the C6orf211 protein (a.a.
• Figure 2 shows that PCNA methyl esterification is promoted by DNA damage, a, Doxorubicin (Dox) promotes PCNA methyl esterification in MCF7 cells, b, PCNA-dependent methyltransferase activity is altered following Dox treatment, c, Dox induces p21 expression in MCF7 cells, d, p21 binding promotes PCNA methyl esterification. e The p21 -induced basic shift is a result of PCNA methyl esterification. f, p21 does not interact with Arml . g, PCNA does not directly interact with Arml in SK-Br-3 cells.
• Dox Doxorubicin
• Dox induces p21 expression in MCF7 cells
• d p21 binding promotes PCNA methyl esterification.
• e The p21 -induced basic shift is a result of PCNA methyl esterification.
• p21 does not interact with Arml .
• PCNA does not directly interact with Arml in
• Figure 3 shows that Arml -dependent PCNA methyl esterification is linked to DNA repair, a, Arml promotes PCNA methyl esterification following DNA damage, b, PCNA-dependent methyltransferase activities are altered in shRNA expressing SK-Br-3 and MCF7 cells, c, DNA damage sensitivity in Arml knockdown cells is related to p53 status, d, Arml knockdown promotes DNA repair.
• Figure 4 shows that Arml promotes DNA damage tolerance, a, PCNA chromatin stability is un affected by Arml . b, Arml is recruited to the chromatin and promotes PCNA ubiquitylation. c, Arml -dependent methyltransferase activity promotes Radl8's interactions with Arml and PCNA. d, Arml interacts with Revl .
• Figure 5 shows a model for PCNA methyl esterification.
• Figure 6 shows results of identification of the C6orf211 protein in the PCNA-dependent carboxyl methyltransferase active fraction, (a). Fractions from passage over a Superdex S200 gel filtration column, (b) Active fractions resolved by 2D-PAGE and stained with colloidal Coomassie Blue. The position of the 50 kDa product of the uncharacterized gene C6orf211 is identified with an arrow, (c) Proteins present in the enriched fractions identified by mass spectrometry and grouped by cellular function.
• Figure 7 shows sequence alignments of CheR, C6orf211, and PIMT proteins.
• Amino acids are colour coded green (polar), red (nonpolar, hydrophobic), pink (basic), and blue (acidic).
• Figure 8 shows alignment of the C6orf211 proteins from eight eukaryotic organisms with motifs I and II and regions II and III identified.
• Figure 9 shows patterns of methyl esterification of peptides from p21-PIP affinity purified PCNA isoforms separated and excised from 2D-PAGE gels and analyzed by LC- MS/MS. Positively identified peptide sequences are shown in black and unobserved sequences are shown in red. The locations of methyl esterified residues in the PCNA isoform spots are presented in bolded blue.
• Figure 10 shows results of lentiviral shRNA knock-down of Arm expression in MCF7 and SK-Br-3 cells, a, shRNA expression, b, reduction of Arml mRNA expression in shRNA expressing cells.
• the invention relates to the treatment of cancer using DNA damaging agents.
• the inventor has surprisingly discovered that knockdown of a previously uncharacterized gene, acidic residue methyltransferase (Arml), improves the ability of cells having a wild-type p53 gene to survive treatment with DNA damaging agents, while causing cells having mutant p53 genes to become more sensitive to killing by DNA damaging agents. Since more than 50% of cancer cell types have mutant p53 genes, while normal proliferating cells have wild type p53 genes, inhibition of Arml increases both the efficacy and selectivity of DNA damaging agents for killing cancer cells.
• Arml acidic residue methyltransferase
• the invention provides a method for treating a mammal with cancer, the method comprising inhibiting in the mammal acidic residue methyltransferase (Arml) in combination with administering to the mammal a DNA damaging agent.
• Arml acidic residue methyltransferase
• Treating a mammal with cancer means causing in the mammal a reduction of signs or symptoms of cancer.
• “Inhibiting acidic residue methyltransferase 1 (Arml)” means reducing the activity and/or expression of Arml.
• Preferred methods of inhibiting Arml include, without limitation, contacting a cancer cell with a small molecule inhibitor of Arml activity, or a dominant negative mutant of Arml, such as an Arml protein with some but not all of its protein- or substrate-interactive domains inactivated or a genetic suppressor element (GSE) that encodes a fragment of the Arml protein, which interferes with the Arml activity.
• GSE genetic suppressor element
• Additional preferred methods include contacting a cell with an inhibitor of Arml gene expression, including without limitation, a short hairpin RNA (shRNA), a small inhibitory RNA (siRNA), an antisense nucleic acid (AS) and a ribozyme.
• a short hairpin RNA shRNA
• siRNA small inhibitory RNA
• AS antisense nucleic acid
• ribozyme a ribozyme
• administering to the mammal a DNA damaging agent means providing the mammal with a DNA damaging agent by any medically acceptable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal.
• compositions of the invention are administered parenterally, e.g., intravenously in a hospital setting.
• administration may preferably be by the oral route.
• Preferred DNA damaging agents include, without limitation, doxorubicin, 6- mercaptopurine, Gemcitabine, Cyclophosphamide, Melphalan, Busulfan,
• Chlorambucil Mitomycin, Cisplatin, Bleomycin, Dectinomycin, Irinotecan and Mitoxantrane.
• “In combination with” means in the course of treating the same disease in the same mammal, and includes inhibiting Arml and administering the DNA damaging agent in any order, including simultaneous administration, as well as any temporally spaced order, for example, from sequentially with one immediately following the other to up to several hours apart.
• the administration of an inhibitor of Arml and DNA damaging agent may be by the same or different routes.
• the compounds and other inhibitors described above may be incorporated into a pharmaceutical formulation.
• Such formulations comprise the compound, which may be in the form of a free acid, salt or prodrug, in a pharmaceutically acceptable diluent, carrier, or excipient.
• Such formulations are well known in the art and are described, e.g., in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.
• compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
• salts refers to salts that retain the desired biological activity of the above-identified compounds and exhibit minimal or no undesired toxicological effects.
• examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid,
• the compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula --NR+Z--, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide,— O-alkyl, toluenesulfonate,
• methylsulfonate, sulfonate, phosphate, or carboxylate such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate.
• the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious toxic effects in the patient treated.
• the effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.
• the invention provides a pharmaceutical formulation comprising an inhibitor of acidic residue methyltransferase (Arml) and a DNA damaging agent.
• Preferred inhibitors of Arml include, without limitation, small molecule inhibitors of Arml activity, dominant negative mutants of Arml, such as an Arml protein with some but not all of its protein- or substrate-interactive domains inactivated, genetic suppressor elements (GSEs) that encodes a fragment of the Arml protein, which interferes with the Arml activity.
• GSEs genetic suppressor elements
• Preferred DNA damaging agents include, without limitation, doxorubicin, 6- mercaptopurine, Gemcitabine, Cyclophosphamide, Melphalan, Busulfan,
• Chlorambucil Mitomycin, Cisplatin, Bleomycin, Dectinomycin, Irinotecan and Mitoxantrane.
• the pharmaceutical formulation may further comprise additional diluents, excipients or carriers, as described above for the first aspect of the invention.
• MCF7 and SK-Br-3 cells were obtained from ATCC and maintained in DMEM or McCoys 5 A supplemented with 10% FBS and antibiotics at 37°C, 5% C0 2 .
• a human Flag-tagged Arml , Revl, and p21 expression construct (Origene) were transiently transfected into SK-Br-3 cells with Fugene 6 (Roche) and extracts generated after 24 h.
• Lentiviral shRNA particles were obtained from Open Biosystems and stably expressing clones selected with puromycin and confirmed by GFP expression and Q-PCR (figure 10). Clonogenic survival assays were performed following exposure to Dox (Sigma) or UV-C using a Spectrolinker (Spectronics) for 4 h. Surviving colonies were stained with methylene blue and counted 2 weeks after treatments.
• the assay was performed as previously described. Cell extracts were assayed with [ 3 H-methyl]-SAM (NEN) for 1 h before equilibration with 100 mM NaOH with 1% SDS and spotting onto filter paper folded into an accordion pleat and placed above scintillation fluid. Diffused H-methanol was detected the following day.
• Sepharose (GE Biosciences). His-tagged human Arml was cloned into a baculovirus expression vector and expressed in Tni insect cells (Allele Biotech., Inc.). GST, GST- p21, GST-p21(PIP), and GST-Radl8 were expressed in BL21(DE3) cells and isolated using glutathione Sepharose (GE Biosciences). GST-p21 was isolated from inclusion
• Anti-Flag immunoprecipitations were performed with anti-Flag M2 Affinity Gel (Sigma).
• p21(PIP)-affinity beads were generated by covalently coupling a synthetic peptide (Anaspec) to CH-Sepharose (GE Biosciences).
• anti-DDK (Flag) antibodies were from Origene
• anti-C6orf211 antibodies were from Sigma
• anti-Radl8 was from ThermoElectron.
• C6orf211 encodes a PCNA-dependent carboxyl methyltransferase (Arml).
• methyltransferase sequences containing motifs I and II and region III were aligned to the full-length C6orf211 protein sequence using KALIGN 39 and align sequences shown. conserveed glycine and glutamic acid residues in CheR motif I and catalytic aspartic acid and conserved isoleucine residues of motif II are underlined. Full-length CheR and C6orf211 protein sequences were aligned with Nomad 40 . CheR's region II sequence is underlined, c, The positions of motifs I, II and regions II and III in CheR and the C6orf211 protein, d, Illustrative representation of the SAM-MT fold in the C-terminus of
• the C-terminus of the C6orf211 protein (a.a. 227- 441) has the potential for a SAM-MT fold. Secondary structures predicted with Jpred 41 were assembled into a hypothetical SAM-MT fold. conserveed a-helices (A-E) are shown in yellow and b-sheets (1-7) are in magenta.
• the CheR structure lacks a-helix C. The positions of motifs I and II in CheR and Arml structures are highlighted in red and blue, respectively, f,
• Recombinant 6 x His-tagged Arml was isolated from Tni insect cell extracts and analyzed by SDS-PAGE and colloidal Coomassie Blue staining, g, Arm possesses a PCNA-dependent carboxyl methyltransferase activity. Purified Arml was assayed in the absence and presence of purified 6xHis-tagged PCNA using the vapour diffusion assay.
• PCNA methyl esterification is promoted by DNA damage.
• Dox Doxorubicin promotes PCNA methyl esterification in MCF7 cells.
• WCE Whole cell extracts
• IB PCNA immunoblotting
• ME -PCNA basic-shifted PCNA isoform
• b PCNA-dependent methyltransferase activity is altered following Dox treatment.
• Dox treated MCF7 WCE were assayed for PCNA-dependent activity in triplicate using the vapour diffusion assay, and average activities are presented ⁇ S.E.M. Significance was determined using a two-tailed t-test.
• Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Flag and anti-Arml antibodies, g, PCNA does not directly interact with Arml in SK-Br-3 cells.
• SK-Br-3 cell extracts expressing Flag (vector control) or Flag- Arml were immunoprecipitated with anti-Flag antibodies.
• Immunoprecipitates were resolved by SDS-PAGE and immunoblotted for Flag and PCNA. Table I. Enrichment of Methyltransferase Activity
• MCF7 (p53 wild-type) and SK-Br-3 (p53- mutant) cells were exposed to increasing levels of Dox and UV and survival determined by clonogenic survival. Average results from three independent experiments are presented ⁇ SD. d, Arml knockdown promotes DNA repair. DNA repair rates were determined in shRNA expressing SK-Br-3 and MCF7 cells using host cell reactivation assay. Cells were transfected with UV -irradiated reporter plasmids and average repair rates were determined after 24 h. Average results from independent experiments are presented ( ⁇ S.E.M.). Significance was determined with a two-tailed t-test.
• Arml is recruited to the chromatin and promotes PCNA ubiquitylation.
• MCF7 cells expressing Arml shRNA were UV -irradiated (20 J/m ) and proteins cross-linked with
• PCNA was incubated in the absence and presence of purified recombinant Arml with and without SAM (10 mM) or sinefungin (20mM) for 1 h at 37°C prior to rocking with glutathione Sepharose bound GST-Radl8 for 15 m at 4°C.
• SAM 10 mM
• sinefungin 20mM
• GST-Radl 8 beads were washed and analyzed by SDS-PAGE and immunoblotting.
• d, Arml interacts with Revl .
• SK-Br-3 cells were transfected with control or Flag-Rev 1 expression plasmids and incubated for 24 h. Cells were harvested 6 h after UV irradiation.
• CheR the C6orf211 gene product, and PIMT were aligned using MUSCLE . Consensus sequences previously determined in CheR and PIMT are shown 13 ' 14 . Amino acids are colour coded green (polar), red (nonpolar, hydrophobic), pink (basic), and blue (acidic).
• the results from this example are shown in Figure 8.
• the C6orf211 proteins from eight eukaryotic organisms were aligned with KALIGN and motifs I and II and regions II and III identified.
• the motif I sequence shows high conservation among all organisms and its location in the primary sequence positions it in the bl/aA loop of the hypothetical SAM- MT fold shown in figure le. conserveed glycine residues of motif I are underlined. Identification of motif II was made using the hypothetical S AM-MT fold.
• the motif II sequence present in the b2/aB loop is also highly conserved among all species. Regions II and III, although less conserved, show significant conservation.
• PCNA PCNA we subsequently enriched for PCNA-dependent carboxyl methyltransferase activity from extracts (figure 6 and table I).
• proteomics techniques then identified proteins comprising the active fractions, and the majority of proteins identified were of known function and were excluded from further consideration. From this approach we were able to rapidly narrow down methyltransferase candidates to an uncharacterized protein, the 50 kDa product of a hypothetical orf on chromosome 6 (C6orf211) . This hypothetical protein was further assessed for methyltransferase potential.
• PIMT isoaspartate methyltransferase
• Arml In addition to PCNA, Arml likely has multiple other targets and it is difficult to speculate as to whether the survival differences observed in the Arml knockdown cells were mediated solely through PCNA methyl esterification.
• an interaction of PCNA with ING1 was previously shown to promote UV-induced apoptosis and prevention of this interaction through either over-expression of p21 or mutation to ING1 's PCNA interacting PIP -box prevented UV-induced apoptosis 36 . It is therefore attractive to postulate that Arml could regulate PCNA's interactions with, among other factors, ING. And loss of Arml 's ability to regulate PCNA's interactions may prevent the cell from effectively responding to DNA damage.
• UV irradiation triggers ubiquitin-dependent degradation of p21(WAFl) to promote DNA repair.
• Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides. J Biol Chem 262, 5622-5629 (1987).

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